Fig. 1.

Experimental strategy for characterization of protein phosphorylation dynamics. A, three SILAC-encoded cell populations were irradiated with 6 grays (6 Gy) and maintained in culture for different lengths of time before the cells were harvested and mixed in a ratio of 1:1:1. Nuclei were purified from the combined cells followed by protein digestion and affinity enrichment of phosphopeptides by ERLIC and TiO₂ chromatography. The resulting phosphopeptides were analyzed by LC-MS/MS, and the data were processed by MaxQuant software and various bioinformatics methods. B, mass spectra of the regulated Nbs1 peptide TTTPGPSLpSQGVSVDEK and the non-regulated GFGpSEEGSR peptide from RNA-binding protein 8A from two separate experiments showing three isotope clusters representing separate time points (o, 0 min; #, 1 h; *, 20 min; +, 8 h; ×, 5 min). C, time profile for the Nbs1 phosphopeptide determined from isotope ratios. D, summary of the identified phosphopeptides.